HPLC DETERMINATION OF VALPROIC ACID IN PLASMA BY CONJUGATION TO A HYDRAZIDE

Abstract

We developed a method to measure plasma levels of valproic acid (VA) using pre-column conjugation of VA to the fluorescent probe, 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH). Swine plasma was spiked with varying concentration of VA. 2-phenylbutyric acid (2PB) was used as an internal standard. VA and 2PB were extracted from plasma (10 µL) using NaCl, MeCN, and ethyl acetate. Extraction efficiency ranged from 85–92%. After conjugation to DCCH, the VA and 2BP, conjugates were easily separated on reverse phase HPLC using a 55–70% MeCN/H2O gradient. Standard curves were generated from the absorbance (λ = 430 nm) and fluorescence (λex = 415 nm, λem = 465 nm) that described an exponential curve y-a(1-bx) with correlation coefficients >0.97. The inter-assay variation measured over 12 days was <3% for the high pool (5 mg/mL) and ≤10% for the low pool (0.5 mg/mL). The intra-assay variation measured over 5 replicates/day was <3.5% for both the high and low pools. The calculated standard curves predicted the actual values of the standards. This assay can be used to measure plasma concentrations of VA in patients or laboratory animals being treated with VA.